HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction Keap1-Nrf2-antioxidant response element (ARE) system regulates cellular defense mechanisms that protect cells from oxidative stress. Indirect inhibitors of Keap1-Nrf2 interaction such as sulforaphane, curcumin, and epigallocatechin gallate are being tested as chemopreventive agents. However, there are general concerns over their use as chemopreventive agents because of their chemical reactivity and their putative irreversible mechanism of action. High throughput screening of chemical libraries is an effective approach to discover small molecule leads as direct reversible inhibitors of protein-protein interactions like Keap1-Nrf2; such direct inhibitors of Keap1-Nrf2 interaction would address concerns over the general use of purified natural thiol- reactive inhibitors as chemopreventive agents. The goal of this current application is to translate our fluorescence polarization (FP) assay to a MLPCN screening center for the screening of MLPCN library. The FP assay detects competitive inhibitors of a fluorescently labeled-Nrf2 peptide from binding to Keap1 kelch domain. Funding for the development of the fluorescent probe and the FP assay has been provided in part by R01 CA133791, Hu L (PI). Secondary and tertiary assays have been established in our labs including an SPR binding assay and a cell-based ARE-luciferase reporter assay. Limited chemical modification will also be performed in the second year to derive initial SAR to enable the selection of hits as leads for future optimization into potent direct inhibitors of Keap1-Nrf2 interaction. Such inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential chemopreventive agents of cancer and other diseases in high risk populations. PUBLIC HEALTH RELEVANCE: Screening of the MLPCN library with our high throughput fluorescence polarization assay will enable the discovery of novel small molecules as direct inhibitors of Keap1-Nrf2 interaction. Specific and potent Keap1- Nrf2 inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential cancer chemopreventive agents in high risk populations.